Abnormal deposition of brain amyloid is a major hallmark of Alzheimer's disease (AD). The toxic extracellular amyloid plaques originating from the aberrant aggregation of beta-amyloid (Aβ) protein are considered to be the major cause of clinical deficits such as memory loss and cognitive impairment. Two-photon excited fluorescence (TPEF) microscopy provides high spatial resolution, minimal invasiveness, and long-term monitoring capability. TPEF imaging of amyloid plaques in AD transgenic mice models has greatly facilitated studies of the AD pathological mechanism. However, the imaging of deep cortical layers is still hampered by the conventional amyloid probes with short excitation/emission wavelength. In this work, we report that a near-infrared (NIR) probe, named CRANAD-3, is far superior for deep in vivo TPEF imaging of brain amyloid in comparison with the commonly used short-wavelength probe. Our findings show that the major interference for TPEF signal of the NIR probe is from the autofluorescence of lipofuscin, the "aging-pigment" in the brain. To eliminate the interference, we characterized the lipofuscin fluorescence in the aged brains of AD mice and found that it has unique broad emission and short lifetime. The lipofuscin signal can be clearly separated from the fluorescence of CRANAD-3 and fluorescent protein via a ratio-based unmixing method. Our results demonstrate the great advantages of NIR probes for in vivo deep-tissue imaging of amyloid plaques in AD.

Fluorescence microscopic imaging (FMI) is one of the fastest growing and most powerful techniques to study cellular activities in a living single cell. FMI has been widely used to monitor the temporal and spatial changes of many important intracellular messengers such as Ca²⁺, H⁺ and cAMP. In the course of our study of cellular responses with confocal scanning fluorescence microscopy, we detected two sources of artifacts which may render experimental observations invalid. First, the water content of the DMSO used could affect the efficiency of loading of the fluorescence indicator into cells and also give rise to spurious fluorescence spots. Secondly, apparently spontaneous temperature-dependent oscillations of BCECF fluorescence and cellular pulsations were recorded in cells which might be misinterpreted as natural rhythmic behavior. These were later shown to be artifacts arising from changes in refractive indices of the immersion oil due to small fluctuations in temperature, which in turn leads to random shifts of the focal plane erroneously manifest as signal oscillations. Based on these observations, certain recommendations for the control and elimination of false images are presented.

The simple diamine diaminoethane (ethylenediamine, EDA) has been shown to activate GABA receptors in the central and peripheral nervous systems, partly by a direct action and partly by releasing endogenous GABA. These effects have been shown to be produced by the complexation of EDA with bicarbonate to form a carbamate. The present work has compared EDA, GABA and β-alanine responses in rat CA1 neurons using extracellular and intracellular recordings, as well as neocortical evoked potentials in vivo. Superfusion of GABA onto hippocampal slices produced depolarisation and a decrease of field epsps, both effects fading rapidly, but showing sensitivity to blockade by bicuculline. EDA produced an initial hyperpolarisation and increase of extracellular field epsp size with no fade and only partial sensitivity to bicuculline, with subsequent depolarisation, while β-alanine produces a much larger underlying hyperpolarisation and increase in fepsps, followed by depolarisation and inhibition of fepsps. The responses to β-alanine, but not GABA or EDA, were blocked by strychnine. In vivo experiments, recording somatosensory evoked potentials, confirmed that EDA produced an initial increase followed by depression, and that this effect was not fully blocked by bicuculline.Overall the results indicate that EDA has actions in addition to the activation of GABA receptors. These actions are not attributable to activation of β-alanine-sensitive glycine receptors, but may involve the activation of sites sensitive to adipic acid, which is structurally equivalent to the dicarbamate of EDA. The results emphasise the complex pharmacology of simple amines in bicarbonate-containing solutions.

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